We have changed the furin site of a model immunotoxin to render it susceptible to cleavage by cancer-expressed proteases including PSA and urokinase. This approach adds another layer of selectivity to immunotoxins besides their binding to surface targets on cancer cells. To construct immunotoxins for treating B-cell malignancies, we produced cDNAs encoding antibody Fd and light chains from an anti-CD19 hybridoma. These clones were used to construct a series of novel single chain immunotoxins. Because their cytotoxic activity was reduced compared to other immunotoxins, construction of improved variants has begun. HIV vaccine: V3 loop vaccines were constructed whereby enzymatically inactive Pseudomonas exotoxin was used as a combination carrier and adjuvant for the delivery of strain specific V3 loops derived from gp120. Rabbits that were injected with such vaccines produced a systemic antibody response that neutralized clinical isolates of HIV-1. Mice that were treated mucosally with the vaccine produced both a systemic IgG response and a salivary IgA response. CaCo2 monolayers, forming tight junctions, were used to show that toxin-V3 loop proteins could transcytose from the apical to the basal lateral side of the epithelia. The entry of the vaccine is via the toxin's surface receptor, LRP. Fragments of chicken LRP were cloned into the LRP-neg cell line, 13-5-- 1, and shown to restore toxin sensitivity to these resistant cells. These studies are being used to identity residues of the receptor that are used for toxin binding and uptake. To promote the entry of telomere- containing plasmid DNA, we have constructed a series of chimeric proteins that are composed of a receptor binding domain joined with TRF. In some constructs, we have incorporated the translocation domain from Pseudomonas exotoxin. When the delivery of telomeric plasmid DNA is optimized, this strategy will be applied to the delivery of high molecular weight DNA vectors containing telomeric DNA.
| Kreitman, Robert J; Stetler-Stevenson, Maryalice; Margulies, Inger et al. (2009) Phase II trial of recombinant immunotoxin RFB4(dsFv)-PE38 (BL22) in patients with hairy cell leukemia. J Clin Oncol 27:2983-90 |
| Pastrana, Diana V; FitzGerald, David J (2006) A nonradioactive, cell-free method for measuring protein synthesis inhibition by Pseudomonas exotoxin. Anal Biochem 353:266-71 |
| Pastan, Ira; Hassan, Raffit; Fitzgerald, David J et al. (2006) Immunotoxin therapy of cancer. Nat Rev Cancer 6:559-65 |
| Hsieh, Jennifer C; Tham, Doris M; Feng, Weijun et al. (2005) Intranasal immunization strategy to impede pilin-mediated binding of Pseudomonas aeruginosa to airway epithelial cells. Infect Immun 73:7705-17 |
| Kreitman, Robert J; Squires, David R; Stetler-Stevenson, Maryalice et al. (2005) Phase I trial of recombinant immunotoxin RFB4(dsFv)-PE38 (BL22) in patients with B-cell malignancies. J Clin Oncol 23:6719-29 |
| Pastrana, Diana V; Hanson, Alison J; Knisely, Jane et al. (2005) LRP 1 B functions as a receptor for Pseudomonas exotoxin. Biochim Biophys Acta 1741:234-9 |
| Wilderman, Paula J; Sowa, Nathaniel A; FitzGerald, David J et al. (2004) Identification of tandem duplicate regulatory small RNAs in Pseudomonas aeruginosa involved in iron homeostasis. Proc Natl Acad Sci U S A 101:9792-7 |
